ACIDC AND ENZYMATIC HYDROLYSIS OF JERUSALEM ARTICHOKE (HELIANTHUS TUBEROSUS L.) TUBERS FOR FURTHER ETHANOL PRODUCTION

Katarzyna Szambelan, Jacek Nowak
Department of Fermentation and Biosynthesis, Institute of Food Technology of Plant Origin, The August Cieszkowski Agricultural University of Poznan, Poland

ABSTRACT

In the presented work we focused on Jerusalem artichoke tubers pre-treatment influence from the point of view of ethanol fermentation efficiency. The influence of pH, acid, temperature and time on hydrolysis yield of the Jerusalem artichoke tubers was investigated. The complete hydrolysis of the Jerusalem artichoke inulin was attained at pH 2.0, adjusted with sulphuric acid, after 1 h at 100°C. The possibility of applying inulinase from Aspergillus niger and commercial invertase to hydrolyse Jerusalem artichoke inulin was also investigated. The results showed that inulinase, in a relatively low dosage, effected 84% hydrolysis of total reducing sugars during time and temperature usually used for fermentation in Polish distilleries (30°C, 72 h). The invertase hydrolysed only 47% of sugars in those conditions.

Jerusalem artichoke tubers might be used as an efficient raw-material in the distillery industry, when commercial yeast or bacteria Zymomonas are used, but the adequate acid pre-treatment of the tuber mash or the addition of the inulinase to the fermentation is necessary.

Key words: acid hydrolysis, enzymatic hydrolysis, ethanol, inulin, Jerusalem artichoke.

INTRODUCTION

Jerusalem artichoke (Helianthus tuberosus L.) has been reported to have one of the highest carbohydrates yield ranging between 5 to 14 t per hectare [12]. The main storage carbohydrate in Jerusalem artichoke tubers is inulin which constitutes 70-90% of all the carbohydrates. Inulin consists of linear polyfructose chains in the β (2 1) position with a terminal glucose residue which is linked to fructose by an α (1 2) bond [8]. Such inulin source as Jerusalem artichoke have recently received much intention as renewable raw material for the production of fuel bioethanol and for remediation of soils contaminated with heavy metals [1, 11, 14]. The ethanol fermentation of Jerusalem artichoke (H. tuberosus L.) tubers is mainly researched with distillery yeast and yeast with inulinase activity or the bacterium Zymomonas mobilis. Microorganisms which have no ability to ferment inulin directly, as distillery yeast or the bacterium, require a hydrolysis step before ethanol fermentation process. The acid hydrolysis of inulin has been investigated utilising mainly sulphuric or hydrochloric acid [3, 4, 10, 11]. The conditions of hydrolysis proposed in these papers varied for pH from 1 to 4, temperature from 60°C to 100°C and the hydrolysis time 5 min to several hours; pH of 3 to 4.5 has been found to give also good results, but pressure above atmosphere was required. Fleming & Wassink [5] applied pH 3-4 and the temperature 70-80°C and achieved 80-100% of inulin hydrolysis but in long time. Toran-Diaz et al. [15] showed that acid hydrolysis of inulin proceeds faster than enzymatic hydrolysis. The authors obtained 73% of total carbohydrates in 1 hour acid hydrolysis comparing to 19% for enzymatic hydrolysis in the same time.

Several enzymes capable of hydrolysing inulin have been described in the literature, originating either from plant material or from microorganisms. The microbial enzymes were obtained mainly from yeast, e.g. Kluyveromyces fragilis, Saccharomyces lactis, Candida kefyr but also fungal inulinases from Aspergillus niger, Fusarium roseum, Penicillum spp. were produced [2, 5, 9, 17]. The microbial enzymes have pH optimum in the range of 3.5-5.5 and at 45-55°C while above 60°C they loose activity rapidly. Some authors [2, 9, 17] demonstrated that fungal enzymes are more effective than the yeast ones. 99% of hydrolysis was achieved in 48 h at 60°C using only 2 units·g^-1, while as many as 6 units·g^-1 of yeast enzymes were needed for 93% hydrolysis in 72 h at 55°C. The enzyme hydrolysis of Jerusalem artichoke inulin was mainly applied for fructose syrups preparation [5, 16].

In this research some parameters of inulin hydrolysis were tested to obtain the highest reducing sugars yield and than high ethanol yield from Jerusalem artichoke tubers fermentation.

MATERIALS AND METHODS

Jerusalem artichoke (H. tuberosus L.) tubers, cultivar Rubik, were obtained from Plant Breeding and Acclimatisation Institute, National Centre for Plant Research in Radzików, Poland. Freshly prepared Jerusalem artichoke mashed tubers were used for experiments.

Commercial inulinase (Sigma-Aldrich, 17 U·g^-1) from Aspergillus niger and invertase preparation (Gaminvert G, 34.500 GIU·g^-1·min^-1) were applied for saccharification.

The sulphuric, hydrochloric and phosphoric acids were used to adjust the pH of the mashed tubers to 2.0, 2.5 and 3.0 for each acid. Than the samples were heated at 80°C and 100°C. The effect of hydrolysis was measured by reducing sugars increase after 30, 60, 90 and 120 minutes for each pH, acid and temperature.

The enzymatic hydrolysis was conducted with inulinase (0.06 mg·g^-1 and 0.12 mg·g^-1 sugars) at pH 5.0 and with invertase (1.79 mg·g^-1 and 17.90 mg·g^-1 sugars) at pH 5.0. There was 30°C applied for enzymatic hydrolysis because that temperature is usually used in ethanol fermentation process both for distillery yeast and Zymomonas bacteria. The degree of hydrolysis was estimated on the basis of reducing sugars content in the mashed tubers after 1, 2, 3, 4, 24, 48, 72 and 96 h.

Polish commercial distillery yeast Bc16a and bacterium Zymomonas mobilis 3881 from Czech Culture Collection, were used for laboratory experiments. The samples were inoculated with yeast or bacterium and incubated at 30°C for 72 h.

Dry matter was determined by drying at 60°C to a constant weight. Reducing sugars were estimated by the Miller’s [7] method using 3,5-dinitrosalicylic acid (DNS), and fructose as a standard. Ethanol was estimated after fermentation by distillation method.

RESULTS AND DISCUSSION

The composition of the Jerusalem artichoke (H. tuberosus L.) tubers, cultivar Rubik, is shown in Table 1. The use of four different parameters: acid, pH, temperature and time, during acid hydrolysis resulted in The different levels of the reducing sugars. Generally, the content of reducing sugars was increasing with time at different acid, pH value and temperature (Table 2 and 3). During laboratory experiments the pH value adjusted with sulphuric acid was proved to give the best results. But at 80°C of the process 2 h were needed to obtain 86% hydrolysis (Table 2). The results obtained with sulphuric acid were 6% and 13% higher (p < 0.05) than those get for hydrochloric and phosphoric acid, respectively. It was also stated that the temperature 80°C was too low to obtain the total hydrolysis of inulin even after 120 minutes, for each of acid or pH value tested.

Therefore, in next part of the research the acid hydrolysis was conducted at 100°C. The effect of acid, pH and time was found significant (p < 0.05) similarly as for 80°C hydrolysis (Table 3). The best results, 196.93 g·kg^-1 of reducing sugars, were observed for pH 2.0 adjusted with sulphuric acid. It was also proved that only 1 hour was needed for complete hydrolysis.

Sulphuric acid at pH 2.0 let obtain more (p < 0.05) sugars comparing to hydrochloric or phosphoric acid, 172.17 g·kg^-1 (87%) and 161.96 g·kg^-1 (82%), respectively (Table 3). Pekić et al. [10] stated that at pH 2.0, corrected with hydrochloric acid, 0.5 h was needed for 95% hydrolysis of artichoke inulin.

The presented results have indicated that in case of all tested acids, the best results (p<0.05) of hydrolysis were observed for pH 2.0 at 100°C in 60 minutes. However, the highest quantity of reducing sugars was obtained using the sulphuric acid as a hydrolysing agent (Figure 1).

Most of the research on acid hydrolysis of Jerusalem artichoke inulin was aimed to the further use of the hydrolysed medium for fructose syrups production [5]. The research concerning the alcohol production from Jerusalem artichoke did not optimalize the acid hydrolysis parameters [11, 15].

The enzymes used in this research were characterised by capability to hydrolyse inulin into fermentable sugars. The enzymatic hydrolysis was conducted at 30°C because the enzymes after hydrolysis (before fermentation process) were not inactivated. That is why they could exhibited activity during fermentation. The long hydrolysis time was caused by lower temperature (30°C) which is not optimum for the action of tested enzymes. But such conditions were applied for further preparation of fermented media.

Inulinase, which has a high activity towards inulin, is so far not present on Polish market as industrial commercial preparation (Fructozyme) and its price is very high. Because of that commercial preparation of invertase (far more cheaper) was also used to check its properties for hydrolysing Jerusalem artichoke inulin.

Testing the influence of the enzyme amount on inulin hydrolysis, it was stated that higher dosages of inulinase or invertase caused important (p < 0.05) increase of reducing sugars content, compared to lower dosages, but only till 24 h of the process (Fig. 2). In the further stage of hydrolysis the addition of higher enzyme amount was not important (p < 0.05). The same correlation showed Guiraud and Galzy [6], who applied inulinase for Jerusalem artichoke juice hydrolysis for fructose syrup preparation.

Therefore, the three-day fermentation process (commonly used in Polish industry) would require relatively low dosage of the enzyme.

Applying inulinase in the hydrolysis process is particularly reasonable for Jerusalem artichoke inulin. The experiments with inulinase (0.06 mg·1g^-1 sugars) let obtain 84% of total reducing sugars (compare to acid hydrolysis) (Table 4). It was also observed that invertase yielded 45% less reducing sugars compared with inulinase (Table 4). Also Fleming and Wassink [5] noticed that while inulinase effected total hydrolysis, invertase effected 15-45% hydrolysis during the same time. The authors worked on fructose syrup production.

Slow rate of carbohydrates decomposition allowed bacterium and distillery yeast to better ferment available sugars (Table 5). Mashes from enzymaticly hydrolysed tubers with inulinase attributed significantly (p < 0.05) higher ethanol yield (84.15% theoretical yield for Z. mobilis and 78.28% theoretical yield for yeast) than those from acid hydrolysed tubers (82.19% theoretical yield for Z. mobilis and 74.36% theoretical yield for yeast) (Table 5).

Results presented in this research and our previous papers [13, 14] proved that Jerusalem artichoke tubers directly (not only juices) might be a very good raw-material for fuel bioethanol production using commercial distillery yeast or Zymomonas bacteria, after adequate pre-treatment by acid hydrolysis (before fermentation) or by the addition of inulinase preparates in the fermentation process.

REFERENCES

1. Antonkiewicz J., Jasiewicz Cz., 2002. Estimation of usefulness of different plant species for phytoremediation of soils contaminated with heavy metals. Acta Sci. Pol. Formatio Circumiectus 1 (1-2), 119-130.

2. Barta J., 1993. Jerusalem artichoke as a multipurpose raw material for food products of high fructose or inulin content. Dep. Canning Technol. Elsev. Sci Publ. B. V.

3. Duvnjak Z., Koren D.W., 1987. Production of fructose syrup by selective removal of glucose from hydrolysed Jerusalem artichoke juice. Biotechnol. Lett. 9, 783-788.

4. Duvnjak Z., Kosaric N., Kliza S., 1982. Production of alcohol from Jerusalem artichoke by yeast. Biotechnol. Bioeng. 24, 2297-2308.

5. Fleming S.E., Wassink J.W.D.G., 1979. Preparation of high fructose syrup from the tubers of the Jerusalem artichoke (Helianthus tuberosus L.). CRC Crit. Rev. Food Sci. Nutr. 1-28.

6. Guiraud J.P., Galzy P., 1981. Enzymatic hydrolysis of plant extracts containing inulin. Enz. Microbiol. Technol. 3, 305-308.

7. Miller G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426-428.

8. Niness K.R., 1999. Inulin and oligofructose: what are they? J. Nutr. 129(07), 1402S-1406.

9. Pandey A., Soccol C.R., Selvakumar P., Soccol V.T., Krieger N., Fontana J.D., 1999. Recent developments in microbial inulinases. Appl. Biochem. Biotechnol. 81, 35-52.

10. Pekić B., Slavica B., Lepojević Z., Petrović S.M., 1985. Effect of pH on the hydrolysis of Jerusalem artichoke inulin. Food Chem. 17, 169-173.

11. Sachs R.M., Viera A.M., Bartolomen M.L., 1981. Fuel alcohol from Jerusalem artichoke. Calif. Agric. 29, 4-6.

12. Swanton C.J., Cavers P.B., Clements D.R., Moore M.J., 1992. The biology of canadian weeds: 101 Helianthus tuberosus L. Can. J. Plant Sci. 72, 1367-1382.

13. Szambelan K., Nowak J., Chrapkowska J.K., 2004. Comparison of bacterial and yeast ethanol fermentation yield from Jerusalem artichoke (Helianthus tuberosus L.) tubers pulp and juices. Acta Sci. Pol., Technol. Aliment. 3, 1, 45-53.

14. Szambelan K., Nowak J., Jeleń H., 2005. The composition of Jerusalem artichoke (Helianthus tuberosus L.) spirits obtained from fermentation with bacteria and yeasts. Eng. Life Sci. 5, 1, 68-71.

15. Toran-Diaz I., Jain V.K., Allais J.J., Baratti J., 1985. Effect of acid or enzymatic hydrolysis on ethanol production by Zymomonas mobilis growing on Jerusalem artichoke juice. Biotechnol. Lett. 7, 527-530.

16. Wenling W., Huiying W.W.L., Shiyuan W., 1999. Continuous preparation of fructose syrups from Jerusalem artichoke tuber using immobilized intercellular inulinase from Kluyveromyces sp. Y-85. Proc. Biochem. 34, 643-646.

17. Zittan L, 1981. Enzymatic hydrolysis of inulin – an alternative way to fructose production. Starch 33, 373-377.

Accepted for print: 29.11.2006

Responses to this article, comments are invited and should be submitted within three months of the publication of the article. If accepted for publication, they will be published in the chapter headed 'Discussions' and hyperlinked to the article.